Cystic fibrosis (CF) is one of the most common lethal genetic diseases among people of European descent, affecting 30,000 individuals in the United States. It is believed that chronic CF lung infections are caused by surface-associated, antimicrobial-resistant communities of microorganisms called biofilms with Pseudomonas aeruginosa being one of the principal pathogens. Current treatment strategies for CF infections, including frequent antibiotic treatment and chest physiotherapy, fail to clear these infections and biofilm bacteria persist in the lung despite intact host immune defenses. Recently, it has been suggested that therapeutic strategies directed towards biofilms may be successful in treating CF lung infections. Our research goal proposed herein is designed to elucidate the nature and identity of proteins that are unique to the biofilm mode of growth for the development of therapeutic strategies directed towards biofilms. Previous work in our laboratories has demonstrated that P. aeruginosa PAO1 undergoes a major shift in its cellular protein profile during biofilm development. This shift is most profound in biofilms grown for 3 and 6 days (maturation-I and maturation-II stage, respectively). We hypothesize that we will identify biofilm-specific proteins - important regulatory, virulence and resistance proteins - that are unique to the maturation-I and maturation-II biofilm stages. We expect that many of the biofilm-specific proteins are post-translational modified and have regulatory functions involved in signal transduction. Our goal will be accomplished by utilizing two-dimensional gel electrophoresis (2D/PAGE) combined with 2D-image analysis and protein identification. Biofilm-specific proteins will be identified by peptide mass fingerprinting using Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS). Upon protein identification, functional proteomics will be used to provide an insight in signal transduction cascades: phosphorylated proteins will be immunoprecipitated and separated by 2D/PAGE. Comparative 2D-image analysis will reveal proteins that are uniquely phosphorylated in the protein patterns of biofilms grown to the maturation-I and -II biofilm stages. Uniquely phosphorylated, biofilm-specific proteins will then be analyzed by peptide mass fingerprinting and MALDI-ToF MS.